Composition for lowering the concentration of intestinal pathogenic peptides

ABSTRACT

The present invention describes a pharmaceutical, veterinary or alimentary composition comprising one or more bacterial strains capable of lowering the concentration of intestinal pathogenic peptides by means of peptidases of probiotic strains. The invention also relates to the use of such compositions, and a method for selection of probiotic strains. Also, the invention relates to novel bacterial strains.

[0001] The present invention relates to a pharmaceutical, veterinary oralimentary composition comprising one or more bacterial strains capableof lowering the concentration of intestinal pathogenic peptides. Theinvention further relates to the use of such compositions for theprevention and/or treatment of a disease or disorders such aspsychiatric disorders, such as autism, ADHD, mood disorder,schizophrenia, pervasive development disorder, bipolar mood disorder anddepression, allergic disorders, celiac disease and multiple sclerosis.The invention also relates to a method for the selection of bacterialstrains, and to novel isolated bacterial strains.

BACKGROUND FOR THE INVENTION

[0002] The etiology and pathogenesis of schizophrenia, autism and themajor mood disorders is still unclear. Genetic factors certainly play animportant role in the development and pathogenesis of these disorders.However, environmental factors contribute, and a combination of the twomost probably operates, as is often seen.

[0003] A typical example of such a combined genetic and dietary baseddisease is Fillings disease, in which a genetic defect in the metabolismof phenylalanin results in severe maldevelopment of the brain. Dietaryreduction of phenylalanine intake, however, prevents the. diseasedevelopment provided it is started shortly after birth. Therefore, anintensive search for environmental factors is going on, the discovery ofwhich could strongly improve therapy of these devastating diseases. Suchenvironmental factors could be infectious or dietary, or even both.

[0004] About 20 ago F. C. Dohan (Dohan, F. C (1983), More on celiacdisease as a model for schizophrenia, Biological psychiatry, 18;561-564;and Dohan, F. C (1988), Genetic hypothesis of idiopathic schizophrenia:its exorphin connection, Schizophrenia Bull, 14:489-494) discussed apossible relation between celiac disease and schizophrenia. Celiacdisease is an inflammatory bowel disorder due to intolerance to peptidesderived from gluten proteins. This condition is occasionally accompaniedby psychiatric and neurological symptoms. A relation between celiacdiseases and psychiatric and neurological disease is further supportedby several recent investigations (KnivsBerg, Ann-Mari, Wiig, Kirsti,Lind, G. Nødland, M., Reichelt, K. L (1990), Dietary Intervention inAutistic Syndromes Brain Dysfunct, 3, 315-327; KnivsBerg, A. M.Reichelt, K. L, G. Nødland, M., Høyen,T (1990), Autistic Syndromes andDiet: a follow-up study, Scandinavian Journal of Educational Research;39, 223-236; Whitley, P., Rodgers, J., Savery, D. and Shattock, p.(1999), An gluten-free diet as an intervention for autism and associateddisorders: perliminary findings., Autism; 3: 45-65; Hadjivassilou, M.,Grunewald, R. A, Chattopadhyay, A. K et al (1998), Clinical,radiological, neurophysiological, and neuropathological characteristicsof gluten ataxia., The Lancet: 352, 1582-1586).

[0005] In 1979, Panksepp (Panksepp, J . A neurochemical theory of autismTrends in Neuroscience 1979;2: 174-177 )proposed the opioid excesstheory in which he suggested that disturbance of endogenous opioid ispart of the pathogenesis in autism. the same time K. L. Reichelt et al (Hole, K., Bergslien, A. A, Jørgensen, H. et al (1979) A peptidecontaining fraction in the urine of schizophrenic patients whichstimulates opiate receptors and inhibits dopamine uptake. Neuroscience;4:1883-1893) isolated biologically active peptides from the urine fromschizophrenic patients. Drysdale (Drysdale, A. , Deacon, R., Lewis, R.et al (1982) A peptide containing fraction of plasma of schizophrenicpatients which binds to opiate receptors and induces hyperactivity inrats Neuroscience; 7: 1567-1574) found a peptide-containing fraction inplasma from schizophrenic patients that were found to bind to opiatereceptors, which induced hyperactivity in rats. Both groups showed thatthe principles they isolated had opioid and dopaminergic activity. Atthe present time, the best-characterized peptides found to be elevatedin psychiatric patients, are exorphins derived from gluten and bovinecaseins.

[0006] Several studies show elevated peptide levels in the urine ofautistic persons (Reichelt, K. L and Teigland-Gjerstad, B (1995)Decreased urinary peptide excretion in schizophrenic patients afterneuroleptic treatment sychiatry Research, 58; 171-176; Shattock, P. andSavery, D.(1997)Evaluation of Urinary Profiles obtained from people withautism and associated disorderes. Part 1 :Classsification of subg0roupshttp://osiris.ac.uk/autism/ps97.htm) The inventors of the presentinvention have confirmed the urinary peptide pattern in normal andautistic individuals, and found higher levels in the autistic children.

[0007] Gluten Derived Exorphins

[0008] During the last year it has become clear that exorphins, a classof biologically active short peptides are produced enzymatically fromgluten proteins in the gut during digestion. (Fukodome et al 1993, 1996,Froetshel 1996). These peptides, which are 4-5 amino acids long, haveopioid activity and are relatively specific for δ-receptors.

[0009] Some representative gluten exorphins have the following aminoacid sequence: Exorphin A5: Gly-Tyr-Tyr-Pro-Thr Exorphin A4:Gly-Tyr-Tyr-Pro Exorphin B5: Tyr-Gly-Gly-Trp-Leu Exorphin B4:Tyr-Gly-Gly-Trp Exorphin C: Tyr-Pro-Ile-Ser-Leu

[0010] DeSantis et al (1997) (DeSantis, A. et al (1997) Schizophrenicsymptoms and SPECT abnormalities in a coeliac patient: regression aftera gluten-free diet, Journal of internal medicine 242:421-423) has shownregression of schizophrenic symptoms and SPECT (Single Photon ComputerTomography) abnormalities in a celiac patient after a gluten-free diet.An involvement of gluten peptides in neurological diseases has recentlybeen shown in celiac ataxia further supporting neuropathological effectsof such substances (Hadjuvassilou et al 1998) (Hadjivassilou, M.,Grunewald, R. A, Chattopadhyay, A. K et al (1998) Clinical,radiological, neurophysiological, and neuropathological characteristicsof gluten ataxia., The Lancet:352, 1582-1586).

[0011] Furthermore, Wakefield 1998 (Wakefield, A. J., Murch, SH,Anthony, A. (1998) Ileal-lymphoid-nodular hyperplasia, non-specificcolitis, and pervasive developmental disorder in children The Lancet351: 637-641 and 2000)( Wakefield, A. J. Anthony A, Murch S H.(2000)Enterocolitis in Children With Developmental Disorders The AmericanJournal of Gastroenerology 95: 2285-2295) has recently found a relationbetween ileal-lymphoid-nodular hyperplasia, non-specific colitis, andpervasive developmental disorder in a group of children. Reichelt et al,1998,( Reichelt, W. H , Ek, J. , Stensrud, M and Reichelt, K. L Peptideexcretion in celiac disease. Journal of Pediatric gastroenterology andNutrition, 1998; 26: 305-309) have demonstrated increased peptideexcretion in celiac disease. Accordingly, the findings of gluten derivedexorphins in the urine of patients with schizophrenia; autism and mooddisorders strongly suggest a causal relation, further supported bydietary experiments.

[0012] The present invention is based on these findings, i.e. that somespecific peptides are found at concentrations above normal, and thatthis is correlated to various disorders or symptoms. In order to preventor treat such diseases it is thus anticipated that compositions capableof lowering this elevated peptide concentration should have an effect onthe disease state.

[0013] Casomorphins

[0014] During the last decade it has also been demonstrated that peptidesequences derived from incomplete catabolism of milk proteins haveopioid activity (Teschemacher, H., Koch, .G., and Brantl, V. (1997) MilkProtein-Derived Opoid Receptor Ligands Biopol. 43: 99-117). Caseinsdegrade to peptides with 3-20 amino acids, some of which have opioidactivity and are termed casomorphins.

[0015] Representative examples of the amino acid sequence of somecasomorphins: β-casomorphin 1-8 Tyr-Pro-Phe-Pro-Gly-Pro- Ile-Proβ-casomorphin 1-7 Tyr-Pro-Phe-Pro-Gly-Pro- Ile β-casomorphin 1-5Tyr-Pro-Phe-Pro-Gly β-casomorphin 1-4 Tyr-Pro-Phe-Pro β-casomorphin 1-4amide Tyr-Pro-Phe-Pro-NH₂

[0016] Recently, Sun et al (1999) (Sun, Z., Cade, J. R , Fregly, M. J.and Privette, R. M. (1999) β-casomorphin induces Fos-likeimmunoreactivity in discrete brain regions relevant to schizophrenia andautism., Autism; 3: 67-83) showed that β-casomorphin 1-7, which is oneof the peptides isolated from the urine from patients with schizophreniaand autism that cause behavioral changes in rats. This peptide alsoinduces Fos-like immunoreactivity in discrete brain regions relevant toschizophrenia and autism (Sun et al 1999).

[0017] Intact peptides can be absorbed from the small bowel. In humansit was demonstrated that bovine casein releases peptides that can passto the blood during digestion of milk or yogurt (Chabance et al, 1998)(Chabance, B. Et al (1998) Casein peptide release and passage to theblood in humans during digestion of milk or yogurt, Biochimie,80:155-165). In normal individuals, however, it appears that thepeptidases in the gut and in the blood degrades such peptides rapidly(Teschemacher, H., Koch, .G., and Brantl, V. (1997)Milk Protein-DerivedOpoid Receptor Ligands Biopol. 43: 99-117). However, this seem not to bethe case for patients suffering of the medical conditions describedabove, where it is believed that such an insufficient degradation orcatabolism of these specific food derived peptides contribute to thedevelopment and severity of such diseases.

[0018] Clinical improvement in autistic children has been demonstratedin clinical trials following exclusion of either gluten or milk and milkproducts form the diet. (Reichelt, et al, 1990, Lucarelli 1995,Knivsberg et al, 1997, Whitley et al, 1999). In addition severalcausistic reports support the effect of gluten free and casein freediets in these patients.

[0019] Further, Singh and Kay reported in 1976 that wheat gluten couldbe a pathogenic factor in schizophrenia, and Reichelt et al (1990) haveshown that a gluten-free diet effects the urinary peptide secretion andclinical state in schizo-phrenic patients.

[0020] Hyperpeptiduria, i.e. increased concentration of peptides in theurine, is regularly found in autism, schizophrenia and major depressivedisorders (Reichelt, W. H, Knivsberg, A. M., Nodland, M., Stensrud, M.and Reichelt, K. L.(1997), Urinary peptide level and patterns inautistic children from seven countries, and the effect of dietaryintervention after 4 years., Dev. Brain Dysfunct ; 10: 44-55; Whitley,P., Rodgers, J., Savery, D. and Shattock, p. (1999), An gluten-free dietas an intervention for autism and associated disorders: perliminaryfindings. Autism; 3: 45-65; Reichelt, K. L., Sagedal, E, Landmark, J.,Sangvik, B. T., Eggen, O., and Scott, H. (1990), The effect ofgluten-free diet on urinary peptide secretion and clinical state inschizophrenia., Journal of Orthomolecular medicine, 5: 223; Reichelt, K.L., Ekrem, J. and Scott, H. (1990), Gluten, milk proteins and autism:dietary intervention effects on behavior and peptide secretion., Journalof applied nutrition; 42: 1-11; Sun, Z. And Cade, J. R (1999), A peptidefound in schizophrenia and autism cause behavioral changes in rats.Autism; 3: 85-95; Wakefield, A. J. et al.(1998), Ileal-lymphoid-nodularhyperplasia, non-specific colitis, and pervasive developmental disorderin children, The Lancet 351: 637-641; Reichelt, K. L and Stensrud, M(1998) Increase in urinary peptides prior to the diagnosis ofschizophrenia, Schizophrenia Research;1998; 34: 211-213). The peptidepattern varies considerably between patients, which may be due to thepresence of dietary peptides without biological effect, and not relatedto enzyme defects in the psychiatric diseases. However, even peptideswithout biological activity may act as peptidase inhibitors. Duringdietary treatment and after use of neuroleptic agents, urinary peptidepatterns are normalized. Other gut-derived substances may also beinvolved, as Shattock has found that indoly-acryloylglycin (IAG) ispresent in the urine of autistic children, and Friedman discloses thepresence of the nonhuman peptide dermorphin.

[0021] These findings indicate that there is a correlation between aconcentration above normal for some specific peptides and thedevelopment of certain diseases, especially neurological disorders.Further, it is clearly established that some of these “pathogenicpeptides” are derived from food proteins, e.g. caseins and gluten.

[0022] Further, there is evidence for genetic alterations in plasmadipeptidyl peptidase IV enzyme activity in depression and schizophrenia(Maes et al, 1994, 1996) (Maes, M., Goossens, F., Scharpe, S. et al(1994) Lower serum prolyl endopeptidase enzyme acivity in majordepression: Further evidence that peptidases play allmenn role in thepathophysiology of depression. Biol. Psychiat.; 35: 545-552 Maes, M.,Goossens, F., Scharpe, S. et al (1994) Lower serum prolyl endopeptidaseenzyme acivity in major depression: Further evidence that peptidasesplay allmenn role in the pathophysiology of depression. Biol. Psychiat.;35: 545-552), which may also affect intestinal enzyme activity.Defective metabolism of gut-derived peptides is reflected in theexcretion pattern of such substances.

[0023] In conclusion, these investigations strongly support the ideathat symptoms in such psychiatric disorders as schizophrenia, ADHD,autism and depressions, at least partly, result from incompletebreakdown and/or increased uptake of gut-derived peptides.

[0024] An object of the present invention is thus to lower theconcentration of these “pathogenic peptides” by increasing the peptidaseactivity in the gut.

[0025] The term “pathogenic peptides” is intended to mean peptidesinvolved in the development, progression or severity of a medicalcondition.

[0026] By the term “peptides” is meant peptides with a sequence of aminoacids, preferable in the range of from 2 to 20 amino acids, and morepreferable less than 10 amino acids.

[0027] It is important to emphasis that these food proteins first aredegraded by proteinases to such peptides, and that another group ofhydrolyzing enzymes, i.e. the peptidases, are responsible for thefurther degradation of these peptides to amino acids which functions asbuilding blocks for protein synthesis.

[0028] A preferred embodiment of the invention relates to the loweringof the concentration of peptides derived from proteins contained in adiet, e.g. the food proteins gluten and casein.

[0029] The basic concept of the present invention is to usemicroorganisms capable of degrading such peptides in the gut.

[0030] A currently preferred embodiment of the present invention relatesto the use of lactic acid bacteria. Preferable these bacteria containpeptidases capable of hydrolysing peptides by adhering to, andcolonizing the gut mucosa. Furthermore, these peptidases may be releasedinto the gut lumen by dead bacteria and thereby degrade unwantedpeptides.

[0031] Biologically active peptides of the exorphin class can be brokendown by various types of peptidases found both in animal cells and inmicroorganisms. In humans peptidases are found in most tissues, butintestinal and blood enzymes may be the most important in relation tothe above-described psychiatric diseases.

[0032] The present invention thus relates to a mixture of one or morebacterial strains capable of degrading all kinds of unwanted peptides inthe gut. A preferred embodiment of the present invention relates to amixture of several bacteria, and where the combined peptidase effectshave a substrate preference towards at least some of the above describedpeptides.

[0033] Lactic Acid Bacterial Peptidase Systems

[0034] Lactic acid bacteria (LAB) inhabit a diverse range ofenvironments such as the gut, various food and beverages such as dairyproducts, meat and wine as well as dead plant and animal materials.These bacteria can colonize the gut, and they play an important role forthe normal gut function.

[0035] Some strains of LAB grow well in milk products, and are usedextensively in the manufacturing of dairy products. Proteins andpeptides can serve as sources for nitrogen, and are metabolized byseveral enzyme systems in the lactic acid bacteria, as reviewed byPritchard and Coolbear (1993) (Pritchard, G. G and Coolbear, T (1993)The physiology and biochemistry of the proteolytic system in lactic acidbacteria FEMS Microbiology Reviews 12: 170-206

[0036] and Yamamoto et al (1993) (angi fullstendige referanse Yamamoto,N. et al (1993) Purification and specificity of a cell-wall-associatedproteinase from Lactobacillus helveticus CP790 J. Biochem, 114:740-745).

[0037] A protein hydrolysing enzyme (protease) is located in thebacterial cell wall of LAB, and is anchored to the plasma membrane.Casein for instance, is split into oligopeptide fragments with 3-20 ormore amino acids by this enzyme.

[0038] Various strains of lactobacilli have different preferences forcaseins, and the proteinases and peptidases of the bacteria havedifferent substrate specificity. The proteinases from L. Lactis subsp.cremoris showed marked preference for δ-casein, while the proteinasesfrom L. Lactis subsp. lactis degrades α- and β-casein.

[0039] The oligopeptide products of the proteinase action are taken upby specific transport systems and further degraded by severalintracellular peptidases into di- and tripeptides and finally into aminoacids. These amino acids are used in the synthesis of new bacterialproteins. This degradation process is quite well described for milkderived proteins, but less is known for the degradation of other dietproteins, e.g. gluten proteins.

[0040] In order to obtain bacteria capable of degrading the harmfulpeptides involved in the psychiatric disorders described above, thepresent inventors have searched for bacteria containing enzymes capableof providing an efficient degradation of these peptides. Such aneffective degradation involves as indicated above several factors;

[0041] 1 ) an efficient uptake system for such peptides, and

[0042] 2) an effective internal catabolism of peptides by severalpeptidases acting together, and

[0043] 3) release of peptidases by dying LAB and the external activityof the bacterial peptidases.

[0044] Since it appears that the enzymatic machinery in the gut is notsufficient to catabolize these short peptides that are involved in thepsychiatric diseases referred to above, a main object of the presentinvention is to provide a composition capable of enhancing the peptidelowering activity, preferable in the intestine system.

[0045] Preferably, these pathogenic peptides should be broken downcompletely to amino acids. As shown in the experimental section we havetested several bacteria for the ability to degrade peptides postulatedto be involved in said diseases. It is thus anticipated that bycombining several 'strains of bacteria, preferably lactic acid bacteriawe will obtain a peptidase mixture capable of eliminating these harmfulpeptides from the gut, and thus to alleviate and/or treat the manifesteddisease condition. Said composition of bacteria can possible also beused as a prophylactic agent for such disorders.

[0046] An object of the present invention is thus to identify acombination of bacteria, preferably LAB's that meets these requirements.The representative bacteria described below are selected from healthyhumans.

[0047] A further object of the present invention is to modulate variousbacterial strains so that they exhibit the desired p'eptidase mixture,and the present invention thus also relates to genetic modifiedmicroorganisms.

[0048] The increased urinary excretion of peptides in psychiatricdiseases appears to depend on an increased uptake from the gut. It isnot known what is the primary cause for this, but a decrease in gutpeptidases activity may explain the increased uptake. Whatever the causeis for the increased uptake of peptides in the gut, one must assume thatthe uptake depends on the level of peptides in the gut lumen. It is thusanticipated that the breakdown of pathogenic peptides in the gut willreduce the peptide uptake, and it is postulated that this will preventand/or treat diseases ca'used by these peptides. Further, this will alsoappear as a reduced excretion of peptides in the urine.

[0049] The lactic acid bacterial peptidases in accordance to the presentinvention must be able to break down peptides such as casomorphins andgluteomorpins. It has been shown that Lactobacillus casei is able tohydrolyze casomorphin 1-7 in vitro by its aminopeptidase IV. Highcontent of peptidases has been shown in various species of Lactobacillushelveticus.

[0050] The' bacteria of the preset invention will be provided as apharmaceutical or nutraceutical composition, and administered to apatient in need of such treatment, preferable as an oral composition oras a supplement agent in food products.

[0051] The bacterial strains must have properties that make them activein the intestinal system, by physically adherence and colonization, andby having efficient uptake mechanisms and high intracellular degradingcapacity, such that the peptides are degraded rapidly in the gut afterfood intake. Release of peptidases to the intestinal lumen may furtherincrease peptide degradation, either from living or dead bacteria.

[0052] A preferred embodiment of the invention provides bacteria thatare capable of recolonizing the gut, at least for a limited period.

[0053] Further, it is essential that the bacterial strains used inaccordance with the present invention are active in the intestinalenvironment that is characterized by low pH and high concentration ofbile.

[0054] The preparations in accordance with the present invention mayalso be used in celiac disease and in allergic disorders, and othernon-neurologic diseases. Both of these conditions involve reactions toforeign peptides and p'roteins. A complete or increased breakdown in thegut will eliminate or impede the uptake of disease promoting peptides,and could be used as a prophylaxis or treatment for the above indicateddisorders.

[0055] A preferred embodiment of the present invention relates to aproduct being a capsule containing 2-4 strains of lyophilized livingbacteria. Further preferred embodiments relates to a fermented milkpreparation and to chewing tablets. The bacteria should have apreference for, and an enhanced degrading activity towards thepathogenic peptides in the gut.

[0056] Preferably, some of the bacterial strains contained in saidpreparation must be able to adhere to the mucosal surface, and grow andbecome part of the intestinal flora, and the product bacteria mustretain their enzymatic activity in vivo.

[0057] Thus, the present invention relates to a pharmaceutical,veterinary or alimentary composition comprising one or more bacterialstrains capable of lowering the concentration of intestinal pathogenicpeptides.

[0058] Preferred embodiments relates to medical disorder or diseasesbehavioral or psychiatric disorders, such as autism, ADHD, mooddisorder, schizophrenia, pervasive development disorder, bipolar mooddisorder and depression, allergic disorders, celiac disease and multiplesclerosis.

[0059] Preferred embodiments relates to composition, wherein thecomposition comprises a Lactobacillus strain selected from the groupcomprising, Lb. helveticus, Lb. acidophilus, Lb. lactis, Lb. casei,streptococcus, bifidobacterium or micrococcus.

[0060] More preferred embodiments relates to bacterial strains selectedfrom the group comprising Lactobacillus crispatus sp, Lactobacillus parapraracasei sp, Lactobacillus fermentum sp, Lactobacillus plantarum spand Lactobacillus acidofilus, and especially to the bacterial strainsare selected from the group comprising NEU 458, NEU 421, NEU 480, NEU401 and NEU 427.

[0061] The present invention also relates to the use of a compositionfor the preparation of a pharmaceutical or nutraceutical composition forthe prevention and/or treatment of a disease or disorder caused ormaintained by an elevated level of a (pathogenic) peptide in theintestine, wherein the composition comprising one or more bacterialstrains capable of lowering the concentration of intestinal pathogenicpeptides.

[0062] Representative diseases or disorders are selected from the groupcomprising behavioral or psychiatric disorders, such as autism, ADHD,mood disorder, schizophrenia, pervasive development disorder, bipolarmood disorder and depression, allergic disorders, celiac disease andmultiple sclerosis.

[0063] The invention also relates to a method for the selection ofbacterial strains capable of suitable for lowering the concentration ofintestinal pathogenic peptides, wherein the various bacteria areselected based on the following steps:

[0064] a) determine the concentration of various peptides in abiological sample, for instance the urine or blood,

[0065] b) determine if some of these peptides are involved as acausative agent in a medical disease or disorder, and

[0066] c) select one or more bacterial strains that have shownpreference for said peptide in an in vitro peptidase assay.

[0067] Further, the invention relates to the isolated bacterial strainsNEU 458 and NEU 421 deposited on Sep. 26, 2002 at the DSMZ under numberDSM 15224 DSM 15223, respectively.

[0068] Experimental Section This section contains experiments thatdemonstrate which properties probiotic microbial organisms need, inorder to break down pathologic peptides in the gut lumen. All probioticorganisms must be safe. Effective probiotic organisms must containpeptidases that are able to hydrolyse the actual peptides withpathologic properties. In order to survive conditions in the stomach andthe intestines they must be able to tolerate acidic conditions and bileacids. In order to have lasting effects they must be able to adhere to,and at least for some time colonise the mucosal surface. In additionmicrobial probiotic strains must grow well in a fermentor for productionreasons.

[0069] The isolated probiotic strains presented in the invention wereselected from bacteria isolated from epithelial cells from theurogential or gastrointestinal system. Cell samples were obtained fromhealthy human donors. Initially the bacterial samples were grownanaerobically on MRS agar plates. Cloned bacteria were sytematicallytested for the properties described elsewere in the specification. Theselected probiotic strains were classified as lactic acid bacteria,since they were gram-positive, produce acid when they were grown, andthey grew in the presence of 0.02% NaN₃. Bacteria that were classifiedas lactic acid bacteria were tested by the comerially available API-testto determine the strain. In this comercial test obtained fromBiomerieux, which describes the fermetation pattern of carbohydratesubstrates, we used the API 50 CH version.

[0070] Bacterial Strains

[0071] All lactic acid bacterial strains were grown from stocks storedat −84° C. (in 30% glycerol). The strains were grown in Man, Rogosa andSharpe (MRS, Merck) broth at 37° C. Before testings the strains werereplated 3 times.

[0072] Radio Labelling for Adhesion Assay

[0073] For metabolic radio labelling 40 μl ³H-adenine was added to a 20ml bacterial suspension in MRS broth and incubated for 16-18 hs. Toremove the excess radiolabel after growth, bacteria were centrifuged(2000 rpm) and the pellet was washed twice with phosphate bufferedsaline (PBS, pH 7,2). The optical density at 600 nm of the bacterialsuspension was adjusted to 1.0 to give approximately between 6×10⁸ and2×10⁹ colony forming units (CFU) ml⁻¹.

[0074] Caco-2 Cell Culture

[0075] The Caco-2 cell line (ATCC CRL-2102) was purchased from theAmerican Type Culture Collection. The cells were cultured in RPMI 1640supplemented with 10% fetal bovine serum and 2 mM L-glutamine and 1%penicillin/streptomycin at 37° C. in an atmosphere of 5% CO₂/95% air.

[0076] For adherence assays Caco-2 cells were grown in 96-well tissueculture plates. Cells were seeded at a concentration of 3×10⁴ cellsml⁻¹(200 μl in each well) and maintained for two weeks prior to use inadherence assays. The cell culture medium was changed every other day,in addition to two hours before adherence assay.

[0077] HEp-2 Cell Culture

[0078] HEp-2 cells were obtained from the ATCC and they were originallyisolated from a patient with a larynx carcinoma. This cell line has beenused extensively as a model in bacterial adherence studies. The cellswere cultured in 75 cm² flasks in Dulbecco's modified Eagle's mediumwith 4 mM L-glutamine, adjusted to contain 4,5 g/L glucose, 1,5 g/Lsodium bicarbonate, 1,0 mM sodium pyruvate, 10% fetal calf serum (FBS)and 1% penicillin/streptomycin at 37° C. in an atmosphere of 5% CO₂/95%air.

[0079] HEp-2 cells were cultured to confluency. The cells weretrypsinated 48 hours before adherence experiments and seeded in 96-wellmicrotiter plates with 200 μl per well from a cell suspension with2.5×10⁵ cells/ML giving 5×10⁴ cells per well.

[0080] In Vitro Adherence Assay

[0081] The adherence of bacterial strains to Hep-2 or Caco-2 cellcultures was examined by adding 200 μl of radio labelled bacterialsuspension to the wells. Before adding the bacteria they were suspendedin MEM-Earle supplemented with 0.5% FBS, 1% L-glutamine and 0.1% NEA,all wells were washed twice with this medium. After incubation for 1-3hours the Caco-2 cell culture were washed with 3×250 μl buffer salinesolution (BSS) in a Multiwash⁺ plate washer (Labsystems) and treatedwith 150 μl of 2% SDS in 0.01 M NaOH for 20 minutes to lyse thebacteria. The lysed bacteria were then mixed with scintillation liquidand the radioactivity was measured by liquid scintillation. The adhesionratio (%) was calculated by comparing the radioactivity of the originalbacterial suspension that was added to the final count of lysed cells.

[0082] Peptidase Activity

[0083] Using pNA labelled substrates, the peptidase activity of variousbacterial strains was tested. The bacteria were harvested in the latelog phase, and used to make a bacterial cell free extract containingpeptidases. The peptidase extract were incubated with the labelledsubstrates, and the reaction was visible as a change in colour. Thepeptidase activity were calculated and given as nmol product per min permg protein

[0084] The bacteria were harvested in the late log phase bycentrifugation at 7500 g at 4° C. for 10 minutes. After washing twicewith 50 mM Hepes pH 7.0, containing 15 mM CaCl₂ to preventautoprotolysis, the bacterial cells were resuspended in 50 mM HEPESbuffer pH 7.0 without calcium. Bacterial cell free extract was obtainedby first freezing and thawing the cells 3 times, then disrupting thecells by sonication at 4° C. for 5 minutes and centrifuged at 15 300 rpmat 4° C. for 10 minutes. The peptidase extract was kept at −20° C.maximally 3 weeks, until it was used in experiments. The amount ofprotein was determined with BCA protein kit at 562 nm.

[0085] The reaction between the substrate and the peptidase took placein 300 μl micro wells (NUNC) at 37° C. The colour change of a mixture of100 μl of substrate and 100 μl of the peptidase extract was followed forwith an automatic microplate reader at 414 nm using the GenesisSoftware.

[0086] Uptake and Metabolism of Peptides by Lactic Acid Bacteria

[0087] The bacteria were harvested in the late log phase bycentrifugation at 7500 g at 4° C. for 10 minutes. Before the transportassays were performed, cells were washed twice with 100 mM MES-KOH(potassium-2-(N-morpholino)-ethanesulphonic acid), 2 mM CaCl₂, pH 6.5(all buffers at a temperate at 4° C.). Cells (A₆₆₀=25) were de-energizedwith 10 mM 2-deoxyglucose for 20 min at 37° C., and washed twice with100 mM MES-KOH, pH 6.5.

[0088] For transport assays, cells (A₆₆₀=10) were pre-incubated for 3min in MES-KOH, with 25 mM glucose, after which 0.5 mM peptide wasadded.

[0089] The solution of peptides and bacteria was incubated at 37° C.Samples were taken after 0, 5, 10, 15, 30, 60 and 120 minutes. The cellswere removed by centrifugation, and the supernatants were analysed usingRP-HPLC.

[0090] The intracellular peptides were extracted, by adding 300 μl of asuspension of 5% perchloric acid and 10 mM Na-EDTA to the pellet, andincubated for 30 minutes. From this suspension, 110 μl was transmittedto an eppendorf tube with 100 μl 1 M KOH-KHCO₃.

[0091] The samples (100 μl) were analysed on an AKTA Purifier fromPharmasia Biotech, with a UV-900 detection unit, using a Vydac 218TP54C-18 column. The peptides were eluted with a gradient from 0-35% B, in100 minutes. Buffer A was 0.1% TFA in water, and buffer B was 0.1% TFAin 60% acetonitrile. The flow rate was 1 ml/min and the peptides weredetected at 215 nm.

[0092] Uptake of Peptides in Caco-2 Cells

[0093] Caco-2 cells were used as an intestinal model system. A 24 wellmicro plate was used. A Lb. acidophilus were added to half of the wells.This is a lactic acid bacterium that adhere to the Caco-2 cells. All thewells were supplemented with β-casomorphine-7, and the concentration ofβ-casomorphine-7 was established at time 0, 60 and 120 minutes afteraddition of the peptide.

[0094] The wells were washed twice with 500 μl 0.5% serum medium withoutantibiotics. All the medium was removed from the wells before thebacteria/medium was added and incubated for one hour at 37° C. in anatmosphere of 5% CO₂/95% air. The wells were washed four times with BSS.The peptide (0.25 mM) was added in a medium without antibiotics andserum

[0095] The samples (100 μl) were analysed on an AKTA Purifier fromPharmasia Biotech.

[0096] Results

[0097] The table I below shows some bacterial stains which can be usedas probiotic stains according to the invention. TABLE I Characterisationof selected probiotic strains by API C50 testing Bacterial strainIdentity by API testing NEU 458 Lactobacillus crispatus sp NEU 421Lactobacillus para praracasei sp NEU 480 Lactobacillus fermentum sp NEU401 Lactobacillus plantarum sp NEU 449 Lactobacillus crispatus sp

[0098] In Vitro Adherence Assay

[0099] Several bacterial strains were tested for the ability toadherence to Caco-2 cell lines. The capabilities of adhesion of somebacteria are given in table II, below. TABLE II Examples of adhesion tocultured Caco-2 cells for some lactic acid bacteria Bacterial strainAdhesion % NEU 458 13 NEU 480 22 NEU 421 44 NEU 449 42 NEU 401 11 NEU427 49 Lactobacillus GG 12 Lb Helveticus AL2 1 Lb Helveticus ATCC 150091

[0100] Adherence experiments using the HEp-2 cells gave similar results,data not shown.

[0101] Peptidase Activity

[0102] Several bacterial strains were tested for peptidase activityagainst the Gly-Pro-pNA substrate, which gives an indication on the PepXactivity. The example of peptidase activity of some bacteria is given intable III, below. TABLE III Examples of peptidase activity for somelactic acid bacteria with various chromgenic substrates Strain SubstrateNEU458 NEU480 NEU421 NEU427 NEU427 NEU449 Helv AL2 Helv 15009Gly-Pro-pNA 50.9 10.5 18.1 7.6 7,6 35.2 30 64 Gly-pNA 8.6 1.6 2.3 0.50,5 4.1 5.7 5.5 Pro-pNA 7.0 0 2.8 2.7 1,9 4.1 5.6 4.2 Phe-Pro-Ala-pNA16.5 0 7.4 4.6 4,6 9.3 15 20 Ala-Phe-Pro-pNA 19.7 2.5 6.0 3.1 3,1 21.710 13

[0103] Transport Assay

[0104] Uptake and degradation of the peptides dermorphin,β-casomorphin-8, β-casomorphim-7 and exorphin A5 have been studied intwo Lb. helveticus bacteria and one Lb. acidophilus. Table IV shows theability of various bacterial strains to degrade some specific peptides.TABLE IV Examples of uptake and degradation of peptides by some lacticacid bacteria. Peptide Bacterial β- β- Exorphin Strain Dermorphincasomorphin 8 casomorphin 7 A5 Lb. 208.7 61.1 24.4 25.6 helveticus ATCC15009 Lb. 386.6 9.6 helveticus AL 2 Lb. 35.2 acidophilus

[0105] Some of the results given in table 3 are summarised in FIGS. 1and 2. FIG. 1 shows the extra cellular degradation of β-casomorphin 8,β-casomorphin 7 and exorphin A5 in Lb. helveticus ATCC 15009. Theconcentration at 0 minute equals 100%.

[0106]Lb. helveticus degrades the three peptides very fast, even thoughthe peptides have different amino acid sequences. This corresponds withthe observation that this bacterium contains several peptidases givingcombined broad substrate specificity (table 3. FIG. 2 shows thedegradation of β-casomorphin 7 and exorphin A5 in Lb. helveticus AL2.The concentration at 0 minute equals 100%.

[0107] This bacterium too had high activity towards the substrateGly-Pro-pNA (table 3): This bacterium has the highest activity towardsthe exorphin A5.

[0108] This examples show that the peptidase content is reflected in theability pf intact cells to metabolise peptides, however, indicating thecomplexity.

[0109] 2-deoxy-glucose was used to empty the bacteria for amino acidsand peptides, and it was anticipated that the intracellular peptidespresent were derived from the supplemented peptides. FIG. 3 shows theintracellular concentration of β-casomorphin 7 and exorphin A5 in Lb.helveticus 300.

[0110] Uptake of Peptides in Caco-2 Cells

[0111] The concentration of β-casomorphin-7 was lowered more quickly inan environment where a Lb. acidophilus was incubated together with theCaco-2 cells than in an environment without these bacteria.

[0112] Table 4 shows the concentration (μM) of β-casomorphin-7 whenCaco-2 cells were incubated with or without Lb. acidophilus. TABLE VConcentration of β-casomorphine-7 incubated with and without Lb.acidophilus. 0 60 120 With Lb. 330.3 117.1 123.7 acidophilus Without Lb.294.4 245.4 171.7 acidophilus

[0113] These results are also shown in FIG. 4, which shows theextracellular concentration of β-casomorfin 7 in the culture medium ofCaco-2 cells with and without an adhered probiotic strain. This findingsupports the idea of the present invention, that probiotic bacteriaadhered to intestinal cells may increase the breakdown of pathologicalpeptides.

[0114] Discussion

[0115] Substantial evidence indicate that diet derived opioid peptidesare involved in the pathogenesis of autism and other psychiatricdiseases, and a reduction of the peptide level in the gastrointestinaltract has been shown to reduce symptoms in such patients. Furthermore,in autistic individuals gastrointestinal symptoms are frequentlyobserved.

[0116] Oral treatment with vancomycin led to an improvement in 8 of 10children indicating a role for some bacterial agent (Sandler RH,Finegold SM Bolte ER et al Short term benefit from oral vancomycintreatment of regressive-onset autism. J Child Neurol, 2000;15: 429-35) .

[0117] In a recent study (Finegold, S M, Molitoris, D, Song, Y et al.Clinical Infectious Diseases 2002; 35 (Suppl 1) S6-16) it was found thatthe gastrointestinal microflora was disturbed in individuals with lateonset autism.

[0118] The high adherence capacity combined with bile tolerance of theprobiotic strains selected according to the present invention willre-establish the microflora in this patient group.

[0119] In order to support the claimed invention a range of lactic acidbacteria were isolated from human epithelial cells. Bacterial strainswere selected based on several criteria such as identity as lactic acidbacteria, adherence to cultured Caco-2 cells, peptidase content, andbile tolerance.

[0120] The strains presented in table I all grew well anaerobically inMRS-medium, and were all gram positive, acid producing and grew in thepresence of azide. They were classified using the APICH50 fermentationsystem.

[0121] A prerequisite for colonisation of a mucosal surface is thecapability to adhere to the cells of the epithelial surface. It is welldocumented that the human epithelial cell line Caco-2 expresses specificintestinal properties such as surface microvilli, enzyme systemstransport mechanisms when cultured for 2 weeks. Thus, this cell line hasbeen used extensively as a model system to study bacterial adherence.The bacterial strains selected in the present invention all show a veryhigh degree of adherence to this cell line ranging from 11-49%. 2comercially available leveticus strains had very low adherence capacity,below 2% and the extensively studied probiotic strain Lactobacilluscasei GG had an adherence capacity of about 12%. All the strainspresented could grow in the presence of bile. We therefore conclude thatthe probiotic strains presented in table I meet the criteria commonlyused to be able to colonise the human gut.

[0122] The objective of the present invention is to create a productwhich can reduce the level of harmful peptides in the gut. Such peptidesare mainly supposed to derive from food proteins such as casein andgluten, but may also originate from microorganisms. Lactic acid bacteriaused in the dairy industry has been extensively studied, but thesebacteria do not colonise the gut, and can therefore not be used as humanprobiotics.

[0123] During the characterisation of strains isolated from humanepithelial cells, we found a large variation in the peptidase contentamong actively adhering strains. Indeed we have selected bacteria withhigh activity of peptidases necessary to hydrolyse peptides of theexorphin type. X-prolyl-dipeptidyl peptiase, PepX, liberates XaaPro(dipeptides were the penultimat amino acid is prolin) dipeptides fromthe N-terminal end of peptidides containing from 3 to 7 amino acids.

[0124] The specific enzyme activities with various chromogenicsubstrates are shown in table III. The values presented in the table aregiven as nmol product produced per minute and mg protein.

[0125] Gly-Pro-pNA reflects the activity of PepX, and as most exorphinsderived form both casein and gluten contain several proline residues,this enzyme therefore will inactivate most of them. The strainspresented in this table all actively hydrolysis this substrate. Gly-pNAliberates singel amino acids from the N-terminal end, and is a measureof PePC activity or PeP N activity. The strains presentetd inTable IIall are able to hydrolyse Gly-pNA.

[0126] Pro-pNA reflects imini prolidase activity. This enzyme splitsXaa-Pro dipeptides, and activates products formed during the action ofPePX. The selected strains have this enzyme activity. The four strainsalso hydrolyse both Phe-Pro-Ala-pNA and Ala-Phe-Pro-pNA, furthersupporting the peptide hydrolysing capacity of the isolated probioticstrains.

[0127] A combination of the lactic acid bacterial strains selected inthis invention contains a peptidase combination able to break down theexorphins efficiently. Furthermore the broad spectrum peptidase activityof this combination of probiotics will be able to hydrolyze a broadrange of food derived peptides, which will include toxic gliadinpeptides causing celiac disease. A recent publication supports this idea(Di Cagno R, De Angelis M, Lavermicocca P, De Vincezi M , Giovannini C,Faccia M , Gobetti M. Proteolysis by sourdough lactic acid bacteria:effects of wheat flour protein fractions and gliadin peptides involvedin human cereal intolerance Appl Environ Microbiol; 68:623-633)

[0128] Enzyme preparations of selected lactobacilli showed hydrolysis ofthe 31-43 fragment of A-gliadin, a toxic peptide for celiac patients.This strongly supports our idea of using live lactic acid bacteria bothcontaining necessary peptidases and being able to colonize the gutmucosa.

[0129] The probiotic strains selected in the present invention mayindeed also be used as ingredients of an enzyme product. Such a productwould be effective by enzymes acting in the gut content, but also bybacterial cell components adhering to the epithelial surface, as it hasbeen found that peptidase activity may also be associated with thebacterial cell wall or membrane.

1. A pharmaceutical, veterinary or alimentary composition comprising oneor more bacterial strains capable of adhering to the intestinal gut andlowering the concentration of intestinal pathogenic peptides, where saidadhesion capability of a bacterial strain is determined as theindividual bacterial strain's capability to adhere to the cell linesCaco-2 or Hep-2. with at least 8% adhesion, and where said peptidelowering effect is measured as a peptidase activity where at least 2.5nmole of a substrate selected from the group of Gly-Pro-pNA, Gly-pNAeller Pro-PNA is converted per min per mg protein.
 2. Compositionaccording to claim 1, wherein said intestinal pathogenic peptides arecausative for symptoms of behavioral or psychiatric disorders, such asautism, ADHD, mood disorder, schizophrenia, pervasive developmentdisorder, bipolar mood disorder and depression.
 3. Composition accordingto claim 1, wherein said intestinal pathogenic peptides are causativeagents for disease or disorders selected from the group comprisingallergic disorders, celiac disease and multiple sclerosis. 4.Composition according to claim 1, wherein said peptide is derived from adietary protein.
 5. Composition according to claim 1, wherein saidpeptide is derived from the intestinal microbial activity. 6.Composition according to claim 1, wherein said peptide is an exorphin.7. Composition according to claim 6, wherein said exorphin is selectedfrom the group comprising: Exorphin A5 (Gly-Tyr-Tyr-Pro-Thr), ExorphinA4 (Gly-Tyr-Tyr-Pro), Exorphin B5 (Tyr-Gly-Gly-Trp-Leu) Exorphin B4(Tyr-Gly-Gly-Trp) and Exorphin C (Tyr-Pro-Ile-Ser-Leu)


8. Composition according to claim 1, wherein said peptide is acasomorphin.
 9. Composition according to claim 8, wherein saidcasomorphin is selected from the group comprising: β-casomorphin 1-7(Tyr-Pro-Phe-Pro-Gly-Pro- Ile), β-casomorphin 1-5 (Tyr-Pro-Phe-Pro-Gly),β-casomorphin 1-4 (Tyr-Pro-Phe-Pro), β-casomorphin 1-4 amide(Tyr-Pro-Phe-Pro-NH₂) β-casomorphin 1-3 amide (Tyr-Pro-Phe-NH₂)β-casomorphin 1-8 amide (Tyr-Pro-Phe-Pro-Gly-Pro- Ile-Pro-NH₂)β-casomorphin 1-8 (Tyr-Pro-Phe-Pro-Gly-Pro- Ile-Pro)


10. Composition according to claim 1, wherein said peptide isdesmorphin, or other peptides of bacterial origin.
 11. Compositionaccording to claim 1, wherein said peptide is hemopectin derived fromhemoglobin.
 12. Composition according to claim 1, wherein one or more ofsaid bacterial strains are a lactic acid bacterial strain. 13.Composition according to claim 12, wherein the composition comprises aLactobacillus strain selected from the group comprising, Lb. helveticus,Lb. acidophilus, Lb. lactis, Lb. casei, streptococcus, bifidobacteriumor micrococcus.
 14. Composition according to claim 1, wherein one ofsaid bacterial strains are selected from the group comprisingLactobacillus crispatus sp, Lactobacillus para praracasei sp,Lactobacillus fermentum sp, Lactobacillus plantarum spand Lactobacillusacidofilus.
 15. Composition according to claim 1, wherein the bacterialstrains are selected from the group comprising NEU 458, NEU 421, NEU480, NEU 401 and NEU
 427. 16. Composition according to claim 1, whereinone of the bacterial strains is NEU
 428. 17. Composition according toclaim 1, wherein one of the bacterial strains is NEU
 421. 18.Composition according to claim 1, wherein the composition comprises oneor several genetic modified bacterial strains.
 19. Composition inaccordance with claim 1, wherein said preparation in admixture containsbacteria with the ability to grow under the physiological situationsthat are met during the transit in the gastrointestinal tract. 20.Composition in accordance with claim 15, wherein at least one of thebacterial strains has the following properties; a) maintain stabilityand exhibit a peptidase activity in the intestinal system, b) resistantto proteolytic degradation c) active at the acidic environment of theintestine, d) resistant to the intestinal bile acids.
 21. Composition inaccordance with claim 15, wherein at least some of the bacteria have theability to colonize the epithelium.
 22. Composition according to claim19, wherein one of said bacterial strains is capable of colonizing andgrowing in the intestinal mucosa.
 23. Composition according to claim 19,wherein one of said bacterial strains has a sustained retention time inthe intestinal system.
 24. Composition according to claim 1, whereinsaid bacterial strains are present in lyophilized form.
 25. Compositionaccording to claim 1, wherein said compositions are in the form ofcapsules, solutions or drinkable suspensions or powder in sachets. 26.Composition according to claim 1, wherein said composition contains from10⁷ to 10⁹ cells of each strain per single dose.
 27. Use of acomposition for the preparation of a pharmaceutical or nutraeuticalcomposition for the prevention and/or treatment of a disease or disordercaused or maintained by an elevated level of a (pathogenic) peptide inthe intestine, wherein the composition comprises one or more bacterialstrains capable of adhering to the intestinal gut and lowering theconcentration of intestinal pathogenic peptides, where said adhesioncapability of a bacterial strain is determined as the individualbacterial strain's capability to adhere to the cell lines Caco-2 orHep-2 with at least 8% adhesion, and where said peptide lowering effectis measured as a peptidase activity where at least 2.5 nmole of asubstrate selected from the group of Gly-Pro-pNA, Gly-pNA eller Pro-PNAis converted per min per mg protein.
 28. Use of a composition inaccordance with claim 23, wherein the disease or disorder is selectedfrom the group comprising behavioral or psychiatric disorders, such asautism, ADHD, mood disorder, schizophrenia, pervasive developmentdisorder, bipolar mood disorder and depression.
 29. Use of a compositionin accordance with claim 28, wherein the disease or disorder is selectedfrom the group comprising the group comprising allergic disorders,celiac disease and multiple sclerosis.
 30. Use of a compositionaccording to claim 28, wherein one of said bacterial strains areselected from the group comprising Lactobacillus crispatus sp,Lactobacillus para praracasei sp, Lactobacillus fermentum sp,Lactobacillus plantarum sp.
 31. Use of a composition according to claim1, wherein the bacterial strains are selected from the group comprisingNEU 458, NEU 421, NEU 480, NEU 401 and NEU
 427. 32. Use of a compositionaccording to claim 28, wherein one of the bacterial strains is NEU 428.33. Use of a composition according to claim 28, wherein one of thebacterial strains is NEU
 421. 34. A method for the selection ofbacterial strains capable of lowering the concentration of intestinalpathogenic peptides, wherein the various bacteria are selected based onthe following steps: a) determine the concentration of various peptidesin a biological sample, for instance the urine or blood, b) determine ifsome of these peptides are involved as a causative agent in a medicaldisease or disorder, and c) select one or more bacterial strains thathave shown preference for said peptide in an in vitro peptidase assay.35. An isolated bacterial strain characterized in that it possesses thecharacteristics of the NEU 458 strain deposited on Sep. 26, 2002 at theDSMZ under number DSM
 15224. 36. An isolated bacterial straincharacterized in that it possesses the characteristics of the NEU 421strain deposited on Sep. 26, 2002 at the DSMZ under number DSM 15223.